This project aims to investigate cellular and molecular mechanisms underlying HIV capture and transmission by dendritic cells (DCs) using an improved humanized mouse model. HIV invades the human body via mucosal surfaces or blood, however it only actively replicates in lymph nodes. It is generally accepted that HIV transmission from invasion site to replicating site depends on DCs, which are antigen presenting cells uniquely potent in initiating T cell immunity. DCs capture antigens in the periphery and migrate to the lymphoid organs where they process and present the antigens to and activate T cells. HIV hijacks these features of DCs for its viral transmission. DCs distribute in the mucosa and blood where HIV invades the human body and are thus the primary target of HIV. In vitro studies show that DCs can bind HIV via multiple receptors, retain the viral infectivity and transmit the virus to T cells that are activated during DC-T cell interaction, thereby amplifying viral productive replication. These studies, together with DCs' superior migration ability, leads to a general idea that HIV exploits DCs as a Trojan horse for entry into the lymph nodes and transmission to T cells, a process yet to be recapitulated in vivo. It is known that HIV can bind to cells through several receptors including CD4 and CCR5. Another fascinating HIV receptor is DC-specific ICAM-grabbing non-integrin (DC-SIGN), a receptor with multiple ligands and thus multiple functions. DC-SIGN, first identified on monocyte-derived DCs, is especially interesting because it not only mediates HIV capture and transmission but also controls DC migration, thus bridging invasion and replication of the virus. It does so by binding different ligands on HIV envelope (gp120), blood vessels (ICAM-2) and T cells (ICAM3) respectively. Importantly, DC-SIGN expression is not homogeneous on all DCs in vivo. It has been shown that microbial stimuli increases DC-SIGN+ DC numbers and DC-SIGN expression level, which suggests that environments with microbial stimuli amplify DC-SIGN mediated antigen capture. Might this, for example, help explain why circumcision, which dramatically reduces the bacterial load in the male genital area is correlated with reduced risk of HIV infection? If DC-SIGN and DCs serve as molecular and cellular links between microbial stimuli and HIV transmission, then it suggests two important questions: (a) Can microbial stimuli affect the HIV binding and the migration of these DCs in vivo? (b) Which DC subsets bind HIV in vivo and do they depend on DC- SIGN for binding and migration? Understanding these dynamic processes could prove crucial to designing strategies to prevent HIV transmission through targeting DC and DC-SIGN. We hypothesize that microbial stimuli increase recruitment and migration of DC-SIGN+ DCs in humans, thereby increasing DC-SIGN mediated HIV binding and transmission. To test this hypothesis, we will perform experiments with the following three specific aims.
Aim 1. Optimize the humanized mouse model and determine reconstitution efficiency and HIV receptor expression in the steady state and in response to microbial stimuli. We will (1.1) determine how transplant of human thymus and fetal liver CD34+ cells, and Flt3L injection affect the reconstitution efficiency of DC and T cells in NSG-SGM3 recipient, (1.2) measure expression of multiple HIV receptors on different DC subsets in different tissues in humanized mice, and (1.3) assess alteration of DC population in response to injection of LPS, a microbial stimulus.
AIM 2. Genetic targeting of DC-SIGN in humanized mouse and its effect on DC development and function. We will (2.1) compare shRNA and CRISPR/CAS9 strategies for their efficiency of transduction and knockdown of DC-SIGN in humanized mice, (2.2) evaluate how DC-SIGN knockdown affects development and migration of DCs and their interaction with T cells in humanized mouse, and (2.3) evaluate the development and migration of DCs and their interaction with T cells in humanized mouse lacking DC-SIGN in response to microbial stimuli.
AIM 3. Determine gp120 binding to multiple DC subsets in vivo with absence and deficiency of DC-SIGN. We will (3.1) determine binding of gp120 to DCs with DC-SIGN presence or deficiency in vitro, (3.2) determine binding of gp120 to distinct DC subsets in vivo in the steady state and in response to LPS, and (3.3) determine how DC-SIGN knockdown affects binding of gp120 to DCs in vivo.
This exploratory study will produce an optimized animal model to study HIV transmission in vivo. Using this mode, we will investigate the role of dendritic cell and its cell surface receptor in HIV transmission. This study will reveal cellular and molecular mechanisms that link HIV transmission to microbial stimuli.
| Lee, Jaeyop; Zhou, Yu Jerry; Ma, Wenji et al. (2017) Lineage specification of human dendritic cells is marked by IRF8 expression in hematopoietic stem cells and multipotent progenitors. Nat Immunol 18:877-888 |
